Method and system for detecting and recording submicron sized particles

ABSTRACT

A system and method for detecting the presence of submicron sized particles in a sample taken from the environment includes a collecting a sample from the environment and purifying and concentrating the submicron particles in a sample based on the size of the particles. The purified and concentrated particles are detected with an apparatus which includes an electrospray assembly having an electrospray capillary, a differential mobility analyzer which receives the output from the capillary, and a condensation particle device for counting the number of particles that pass through the differential mobility analyzer. The system is intended to collect a sample containing submicron size particles having a size range of from greater than 350 nanometers to about 1000 nanometers and wherein the particles include viruses, prions, viral subunits, viral cores of delipidated viruses, plant viruses, standard particles used for calibrating equipment, coated particles, spherical particles, metallic-core shelled particles, polymers, fluorescent microspheres, powders, nanoclusters, particles produced as a result of manufacturing processes, and portions of bacteria.

COPENDING APPLICATIONS

This application is a Continuation In Part of U.S. patent applicationSer. No. 09/662,788 filed on Sep. 15, 2000, and issue as U.S. Pat. No.6,491,872.

GOVERNMENT INTEREST

The invention described herein may be manufactured, licensed, and usedby or for the United States Government. The invention also relates toU.S. Pat. Nos. 6,051,189 and 6,485,686, assigned to the United StatesGovernment and herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the detection, identification andmonitoring of submicron size particles. More particularly, the inventionpertains to apparatus and methods for the sampling, measuring,characterizing, automated detection, identification, and monitoring ofsubmicron size particles. Preferably, the present invention provides forthe sampling, detection and identification of submicron size particleshaving a size range of from about 5 to about 1000 nanometers. Suchparticles include viruses and virus-like agents (such as, for example,prions, viral subunits, viral cores of delipidated viruses, plantviruses, etc.) in bioaerosols and fluids. Further examples includestandard particles used for calibrating equipment, coated particles,metallic-core shelled particles, polymers, fluorescent microspheres,powders, nanoclusters, particles produced as a result of manufacturingprocesses, and other chemical and biological materials such as segmentednanometer size portions of bacteria.

2. Fields of Use of the Invention

Detection and identification of viruses without limiting the detectionand identification to a particular family, genus and species andsearching for viruses pathogenic to humans in a single environment isdifficult.

The difficulty of detecting and monitoring a wide range of viruses alsovaries by environment, but perhaps a most troublesome environmentinvolves combat conditions, such as a potential biological warfare (BW)threat environment. Notwithstanding the variation in virulence fromvirus to virus, in general the ingestion of 10⁴ virions constitutes asignificant threat to a soldier who breathes on the order of 1,000liters (1 m³) of air per hour. Instruments are needed with sensitivitieswhich enable detection of remote releases of biological agents in afield environment thereby providing early warning capabilities, allowingcalculations for troop movements and wind patterns.

Additionally, it has been difficult to maintain a broad-spectrum systemfor the detection of viruses which are free from false negatives becauseof natural or artificial mutations. Consideration should be given to thehigh mutation rates of known viruses, the emergence of new viruses, suchas the Ebola virus, and the potential for deliberate artificialmutations of viruses. Furthermore, there are virus-like infectiousagents, such as prions, which are suspected of causing scrapie, “mad-cowdisease” and Creutzfeldt-Jakob disease. These prions possess no DNA orRNA, and can withstand 8 MRads of ionizing radiation before losinginfectiousness. Other virus-like infectious agents, such as satellites,possess no proteins.

In the detection and monitoring of viruses recognition should be givento false positives associated with background materials. Backgroundincludes biological debris which obscures the detection of the virusesby registering as a virus when a sample is analyzed. Analysis of virusesrequires a very high degree of purification of those viruses to overcomebackground loading in order to avoid false positives. For example, a BWvirus may be buried within loadings of other microorganisms which formbiological debris having loading on a magnitude of 10¹⁰ larger than thethreshold loading for the targeted virus itself.

Although methods that culture viruses can often be used to increase thevirus over background, culture methods may be too slow for effectiveviral BW detection; furthermore, some important viruses cannot be easilycultured.

As set forth in U.S. Pat. Nos. 6,051,189 and 6,485,686 and U.S. patentapplication Ser. No. 09/662,788 filed on Sep. 15, 2000, assigned to theU.S. Government and herein incorporated by reference, viruses may alsobe extracted from an environment and concentrated to an extent thatpermits detection and monitoring of viruses, without culturingprocedures. Generally, in the detection of small amounts of viruses inenvironmental or biological liquids, it is necessary to both enrich theconcentration of viruses many orders of magnitude (i.e., greatly reducethe volume of liquid solubilizing the viruses) and accomplish removal ofnon-viral impurities. In the presence of non-viral impurities, even themost sensitive detection methods generally require virus concentrationson the order of 10 femtomoles/microliter or more in the sampled liquidto reliably detect the viruses.

Sampling for airborne viruses is generally accomplished by collectingairborne particles in liquid, using a process such as air scrubbing, oreluting from filter paper collectors into a liquid medium. Collectionand subsequent separation and detection methods are affected by theadsorption of viruses into solids in aerosols and liquids.

In contrast, when sampling liquids for viruses, in many cases no specialequipment or processes may be necessary in order to collect a sample;for example, in sampling blood and other body fluids for viruses, only astandard clinical hypodermic needle may be needed. For sampling ofbodies of water or other conveniently accessible liquids, samplecollection may not be an issue at all, and in such cases the term“collector” is often applied to what is, in reality, a virus extractionstep (such as collection on a filter).

Rapid detection translates into protection for soldiers, more reliableand simplified strategic planning, and validation of other BWcountermeasures. Previously known detection methods using biochemicalreagents may often be impractical in the field, even for trainedvirologists. Additionally, reagent-intensive approaches, such asmultiplex PCR, low-strigency nucleic acid hybridization, and polyclonalantibodies, may increase the incidence of false positives severalhundred-fold, whether under highly idealized laboratory conditions or inthe field. Additionally, the hypervariability, or rapid mutation, ofviruses and emergence of new, uncatalogued viruses may preclude methodsbased on biochemical assays, such as PCR, immunoassay, and the like,from achieving broad-spectrum detection of all viruses regardless ofidentity, known or unknown, sequenced or unsequenced.

With respect to nano-size particles having a size range of from about 5to about 1000 nanometers, the ability to manufacture virus-size ornano-size particles has resulted in the commercialization of newprocessing technologies and potential applications of nanoparticles.Likewise, particles produced under controlled manufacturing conditionsmay contain nano-size contaminants. Generally, for example, nano orultrafine powders made of a wide range of metallic, non-metallic,ceramic and semiconducter materials with particle sizes as small as 5 nmare examples of nano-size particles. Mechanochemical processingtechnologies may, for example, use a conventional milling process andinduce specific solid-state reactions to uniquely form separatednano-particles. Nano-size particles may also have optical propertieswith a wide application in the creation of new transparent opticalpapers and film production. Uniform particle size distribution canresult in uniform film quality and small particle sizes that can lead toenhanced resolution. Nano-size particles also have application ascoatings and microchip manufacturing. Nanometer crystallites may consistof organically functionalized, catalytically active metals such asplatinum, palladium and silver or non-active noble metals such as gold.In catalytic processes such particles have extremely high surface areasand size-dependent chemical behavior. In these advanced materials theelectronic, thermodynamic and chemical properties frequently depend upontheir size, shape and surface composition for functionality. It is amajor challenge to control the particle size, morphology and surfacecomposition. It is also a major challenge to properly characterize theparticle size and morphology after the particles are manufactured. Anear real time ability to measure and characterize these particles wouldbe helpful.

Another example of nano-size manufacturing is the production ofultrafine metal particles by evaporation of a metal from a liquid pool,entraining the metal atoms in a hot inert gas carrier and then rapidlymixing in a cold inert gas to cause nucleation and growth. Nearlyuniform sizes with controlled diameters in the nanometer range have beenproduced using this method. Such particles are collected by expandingthe gas stream and impinging the particles onto a surface or byscrubbing the particles out of the gas stream with a liquid spraycontaining a surfactant and collect them as a stable colloid. Thismethod has been used to take advantage of the unique electrical andoptical properties of the nanoparticles, as well as, the using processesto deposit nanometer particles for making ultrasmooth surfaces andmirrors. Nanoparticles have also been made from sodium/halide flames.SEM (scanning electron micrography) images have shown hexagonal andcubic nanoscale (40–50 nm) particles of tungsten-titanium composites.This method prevents agglomeration by allowing nucleation and growth ofthe particles until they reach a desired size and then coat them with anappropriate material before they agglomerate. In this way encapsulatedcore nanometer particles can be produced. The coating material isremoved by heating under a vacuum to produce a resulting powder. Thebenefit of encapsulation can be to narrow the size distribution of thecore particles and thus improve the particle properties.

New methods for depositing nanometer-size thin coatings onto tinyparticles are being considered for use in a wide range of applicationsthat include the manufacturing of safe and more convenient medicinessuch as used in asthma therapy. Pulsed laser deposition techniques havebeen used to coat glucocorticoids, which are a component of asthmatreatments, with thin layers of a biodegradable polymer. Such coatingsare thought to improve the rate of drug release and improve overallblood concentration. A method to measure the increase in the diameter ofthe nanoparticles after coating would be helpful.

The uses and application of nanoparticles is rapidly expanding. Ceramicnanoparticles may provide better resistance to scratching and corrosionof paints and coatings. Improved manufacturing of nanoparticles couldlead to improved catalysts thereby leading to new and betterpharmaceuticals and materials. Batteries may generate more power as aresult of the increased surface areas with metallic or iron-polymers.

To adequately measure and characterize nano-size particles,scanning-probe microscopes with supersharp tips, nanomanipulators,nanotubes, inorganic-organic hybrids and smaller electronics and otheradvances have produced requirements to adequately measure andcharacterize these particles.

For example, one means for counting, measuring and characterizingnanometer particles is with the use of nano-size polystyrene particlesthat are used as size-markers for measuring the dimensions of biologicalstructures. These particles are available from companies such as BangsLaboratories, Inc. for several standard sizes. The company generallysizes a particle three times and reports the average of the results. Afrequently used method to measure nanometer size particles is lightscattering technology, which yields a nominal mean diameter with acoefficient of variation.

Nano-size contaminants have been found in manufacturing processes.Nanometer particles have been discovered in amorphous films of siliconand hydrogen for the use in solar panels by use of a scanning tunnelingmicroscope. These nanometer particles (3–5 nm) were thought to form inthe vapor and bond with the film. They degrade the ability of the filmto convert light into electrical energy. Measuring these nano particlesand characterizing their distribution could help determine a way to keepthe particles from forming or reaching the film surface and thus improvethe films.

Bacteria are completely different types of microorganisms than theviruses. Viruses are a magnitude smaller in size than bacteria. Bacteriaare classified in their own scheme. They have cell walls or areorganized into cellular components and generally are considered to beamong the self-sustaining organisims. Viruses require a living cell toinvade in their life cycle. The technology and processes disclosed inU.S. Pat. Nos. 6,051,189 and 6,485,686 and the abovementioned co-pendingapplication capitalize on the size and physical properties of theviruses to separate, count and characterize them. There is sufficientinformation from this characterization to identify them and performinvestigative studies. Bacteria are generally 0.5–1 microns wide and 2–3microns long, and generally outside the physical ability of theapparatus disclosed in referenced U.S. Patents. Bacteria have, however,interesting features that are in the proper size range for apparatusdisclosed in referenced U.S. Patents to characterize. For example,gram-negative bacteria, named because of their inability to retaincrystal violet-iodine complex stain, have rigid surface appendagescalled “pili.” These “hair-like” structures are around 7 nm in diameterand vary in length, up to 25 nm for the longer flagellae, which areother nanometer-sized structures that can be attached to the surface ofbacteria. The Pili are composed of structural protein sub-units called“pilins.” Some structures have only one structural protein unit, otherPili are more complex and have several. These Pili consist of a precisehelical arrangement of one or more types of protein and as indicated mayhave different lengths for different bacteria. Choudhury, et.al (1999):Science 7 Aug. 1999 285:1061 and David Eisenberg: How chaperones protectvirgin proteins. (Science 13 Aug 99 285:1021), discuss crystal complexesassociated with pilin subunits. Cell lysis breaks the cell intocomponents. Lysis can be achieved by changes in pH, temperature, sonictreatment or by chemical means. The optimum means for releasing the piliproteins has not been well established, but their organization andstructure indicates that controlled heating over a range of 63–70C willfacilitate release. The pili can then been treated as any nanometerparticle, separated and counted. Different pili proteins for differentbacteria species can be expected. Evidence in this manner suggests thatIVDS can indeed see these virus-sized bacteria components and in thismanner detect bacteria. Pili are also found on gram positive rods andround or cocci bacteria.

SUMMARY OF THE INVENTION

A system and method for detecting the presence of submicron sizedparticles having a size range of from about 5 to about 1000 nanometersin a sample taken from the environment. The system includes a collectingmeans for collecting a sample from the environment and a means forpurifying and concentrating or separating the submicron particles in asample by purifying and concentrating or separating the particles basedon size. The purifying and concentrating or separating means includes ameans for connecting the collecting means to the purifying andconcentrating means for transferring the sample from the collectingmeans to the means for purifying and concentrating the particles. Thesystem also includes a means for detecting the purified and concentratedor separated particles, wherein the detecting means comprises: anelectrospray assembly, the assembly having an electrospray capillarywhich receives the output from the purifying and concentrating means, adifferential mobility analyzer which receives the output from thecapillary, and a condensation particle device for counting the number ofparticles that pass through the differential mobility analyzer. Abiomarker means or other calibrating means may be mixed with the sampleand utilized to correlate the results with the known size orconcentration of the biomarker or calibrating means.

The collecting means comprises an ultracentrifuge for density-gradientultracentifugation so that the particles are banded according todensity, or a collector having means for liquid scrubbing a collectedfluid sample of aerosol and gaseous materials containing the particlesand a means for reducing the size of solid materials in the fluidsample. The collecting means may also comprise a liquid samplecollector. The collecting means is intended to collect a samplecontaining submicron size particles having a size from about 5 to about1000 nanometers and are selected from the group consisting of viruses,prions, macromolecules, proteins and satellites, viral subunits, viralcores of delipidated viruses, and plant viruses. Further examplesinclude standard particles used for calibrating equipment, coatedparticles, spherical particles, metallic-core shelled particles,polymers, fluorescent microspheres, powders, nanoclusters, particlesproduced as a result of manufacturing processes, and other chemical andbiological materials such as nanometer size portions of bacteria

The system also includes a means for detecting the purified andconcentrated particles, wherein the detecting means comprises: anelectrospray assembly, the assembly having an electrospray capillarywhich receives the output from the purifying and concentrating means; adifferential mobility analyzer which receives the output from saidcapillary; and which receives the output from the capillary, and acondensation particle device for counting the number of particles thatpass through the differential mobility analyzer. Automated control meanscan be utilized to control the flow of sample through the system.

The method for detecting the presence of submicron sized particles in asample taken from the environment, includes the steps of collecting asample from the environment, purifying and concentrating the submicronsize particles in the sample based on size; and detecting the purifiedand concentrated particles with a detecting means comprising anelectrospray assembly which has an electrospray capillary which receivesthe output from the purifying and concentrating means, a differentialmobility analyzer which receives the output from the capillary, and acondensation particle device for counting the number of particles thatpass through the differential mobility analyzer.

Accordingly, an object of the present invention is to detect known andunknown or submicron size particles.

Another object of the present invention is to provide a method andapparatus for the efficient and rapid detection and identification ofsubmicron size particles based on the physical characteristics of theparticles.

A further object of the present invention to provide an automated systemfor the detection and identification of submicron size particles.

These, together with still other objects of the invention, along withthe various features which characterize the invention, are pointed outwith particularity in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Other objects and advantages of the invention will become apparent uponreading the following detailed description with reference to theattached drawings, wherein:

FIG. 1 is a schematic illustration of the integrated virus detectionsystem of the present invention;

FIGS. 2A and 2B each compare DMA-CNC data for a typical sample beforeand after ultrafiltration, where the solid curve shows data beforeultrafiltration and the dashed curve shows data after ultrafiltration;

FIG. 3 shows a plot of the GEMMA data for two ultrafiltered samples;.

FIG. 4 shows a plot of GEMMA data for MS2 phage for four runs of a knownstandard sample after ultrafiltration;

FIG. 5 shows peak amplitudes and areas plotted as a function ofsuccessive dilutions of the sample of FIG. 4.;

FIG. 6 is a plot of the virus window showing the densities andsedimentation coefficients for viruses pathogenic to man;

FIG. 7 provides a table giving the densities (g/ml) and size (nm) forknown viral families containing species pathogenic to man;

FIG. 8 is one embodiment of the virus detection system;

FIG. 9 is a first ultrafiltration module;

FIG. 10 is a cross-sectional representation of a filter element;

FIGS. 11A and 11B are other embodiments of a virus detection system;

FIG. 12 is a graph of MS2 Bacteriophage with growth media;

FIG. 13 are results of the MS2 Bacteriophage with growth media afterultrafiltration;

FIG. 14 is a graph of MS2 Bacteriophage with albumin;

FIG. 15 are results of MS2 Bacteriophage with albumin afterultrafiltration;

FIG. 16 is a graph of MS2 Bacteriophage with cesium chloride;

FIG. 17 are results of MS2 Bacteriophage with cesium chloride afterultrafiltration;

FIG. 18 are results of MS2 filtered with a 1M centrifuge filter;

FIG. 19 are results of MS2 filtered with a 300K centrifuge filter;

FIG. 20 are results of MS2 in the filtrate;

FIG. 21 is a logarithmic curve for variable dilutions of MS2;

FIG. 22 are results of a GEMMA analysis of MS2 sample, DPM14;

FIG. 23 are results of a GEMMA analysis of MS2 sample, DPM13;

FIG. 24 are results of a GEMMA analysis of MS2 sample, DPM12;

FIG. 25 are results of a GEMMA analysis of MS2 sample, DPM11;

FIG. 26 are results of a GEMMA analysis of a fifth MS2 sample, DPM10;

FIG. 27 are results of a GEMMA analysis of a sixth MS2 sample, DPM9; and

FIG. 28 are results of a GEMMA analysis of a seventh MS2 sample, DPM8.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention comprises a method and apparatus for the detectionand monitoring of submicron particles. The method and apparatus allowsfor the collection, concentration, purification and detection of virusesthat are pathogenic to humans.

As set forth in U.S. Pat. Nos. 6,051,189 and 6,485,686 and as shown inFIG. 1, an integrated virus detection system (IVDS) 100 includes acollection stage 101, an extraction stage 102, apurification/concentration stage 103, and a detection stage 104.

In the collection stage 101, a collector 1 is used for aerosol orgaseous fluid sampling. In aerosol sampling, the collector 1 samplesairborne particles in the approximate size range of from about 2 toabout 10 microns and which may carry viruses and virus-like particleshaving a size range of from about 10 to about 1000 nanometers. Normalcollection rates would be from about 100 to several thousand liters/minof air. Collection of the submicron size virus particles in thecollector 1 is facilitated by the fact that airborne viruses generallytravel in or on aerosol particles which measure larger than a micron. Inexceptional cases where the virus is not rafting on a supermicronfomite, the danger of transmission by inhalation is generally reducedbecause of the distribution of submicron particles in the atmosphere andthe difficulty in capture by the lungs. The collector 1 has a waterinlet 2 which is connected to a water source, such as tap water or awater purification system. The collector 1 scrubs the collectedparticles with the incoming water from the water inlet 2. Examples ofthe collector 1 are the U.S. Army's XM2 or the SPINCON collector made byMidwest Research Institute.

In many applications other than aerosol sampling, samples which maycontain viruses, for example, are obtained without need for what wouldbe considered a formal “collection stage”, such as when the samples arealready in the liquid form. These include, for example, blood samples,obtained by ordinary means familiar in clinical settings, as well asother body fluids such as mucus, semen, feces, lymph, saliva, etc. Alsoin this category are situations involving sampling of bodies of watersuch as municipal water supplies, rivers and lakes, beverages, andhigh-purity water used for microelectronics manufacture.

The collector 1 includes tubing 3 which connects the collector 1 to aholding tank 6 containing a blender or homogenizer 5. The collector 1has an aqueous stream output on the order of 1–10 ml/minute containingthe scrubbed particles which is pumped through the tubing 3, preferablyof TEFLON or polysiloxane-coated to reduce adsorptive losses. The tubing3 is connected to a one liter holding tank 6. Alternatively, the tubing3 can be connected directly to the extraction stage 102 or according toone embodiment of the present invention, directly to a detection stage104.

In the holding tank 6, solids in the aqueous stream are broken up byusing the homogenizer 5, or alternatively, by forcing the aqueous streamthrough an orifice. The homogenizer 5 has a bladed section 34.Surfactant or amphiphile is added at the inlet 4, which preferably ismixed with water prior to entry into the holding tank 6. The surfactantor amphiphile breaks down the structures in the aqueous stream.Preferably, the amphiphile has a low boiling point, which allows easyremoval of the amphiphile in a later stage. Most preferred, theamphiphile is diethylene glycol monohexyl ether. Base is also preferablyadded to increase the pH of the homogenized liquid which tends todecrease aggregation. Examples of the homogenizer 5 are the LightninClosed Tank Model general purpose stirrer model G2S05R, avai lable fromLightnin, a unit of General Signal of Avon, N.Y., catalog no. 869435, orthe PC-controllable stirring motor, RE162 analog, ID no. 8000700 androtor-stator S 50 N-W 65 SK, ID no. 8005100 from IKA Works, Inc. ofCincinnati, Ohio, which serves as part 34.

In leaving the holding tank 6, the aqueous stream passes a screen filter7 which regulates the output of the holding tank 6. The screen filter 7is preferably 10 micron mesh and made of stainless steel or othercorrosion-free material. A pump 8, which is designed for pumping liquidsthrough the tank 6, draws the aqueous stream from the holding tank 6 andthrough the screen filter 7.

Beyond the pump 8, a three-position PC-controlled switch 10 is used toallow the discharge from pump 8 to flow into a centrifuge rotor 12 in afirst position. To understand the function of the second and thirdpositions of this switch, it is necessary to realize that aftercentrifugation, the gradient imprisoned in the rotor can be divided intotwo parts: the useful part which contains that range (or in some cases,those ranges) of densities in which the particles to be detected areexpected to lie, and the remainder which will generally be discarded andnot sent on to the next stage. Thus, for example, in the detection ofviruses pathogenic to humans, this useful part could be that part of thegradient corresponding to densities of 1.175–1.46 g/ml, as discussedelsewhere herein; alternatively, a subset of this range could constitutethe useful range if only certain viruses are being analyzed for.

Thus, the second position of switch 10 allows the useful part of thegradient to flow on to part 30 (in particular, to the first position ofpart 30, as discussed below), and the third position of the switchallows the discarded portion of the gradient from the rotor 12 to flowout through a port 9; if desired, port 9 can incorporate means torecycle density gradient material, if desired. In the first position, asthe screen-filtered sample from the pump 8 travels past the switch 10,it enters into the extraction stage 102.

In the extraction stage 102, the aqueous stream enters a liquid-cooledcoaxial seal 11. After passing the coaxial seal 11, the aqueous streamenters at the upper shaft of the rotor 12. The rotor 12 is a zonalultracentrifuge rotor, such as a Beckman's CF-32 rotor or Z-60 rotor,which is inserted into and spun by a centrifuge 35, such as a BeckmanOptima XL-100K Preparative Ultracentrifuge. For large sample volumeswith small quantities of viruses, for example monitoring of bodies ofwater, such as drinking water sources, the present invention preferablyuses continuous-flow density gradient ultracentrifugation, using forexample the Beckman's CF-32 rotor. For other applications, ordinaryzonal centrifugation is preferred with rotor 12 being a Beckman's Z-60rotor. In a special seal and bearing assembly, fluid inlet and outletstreams access an annular space 13 between a core 32 and rotor wall 33through the coaxial seal assembly II and via port 50. Density gradientsolutions, sample liquid, and the displacement fluid are sequentiallypumped into the annular space 13. Density gradient solutions are loadedfrom port 15 through inlet 14. From pump 8, sample liquid is added. Adensity gradient solution is any liquid which permit the separation ofviruses, such as a sucrose or, preferably, cesium chloride solution.

In continuous flow operation, the virus-containing liquid stream ispumped in from the collection stage 101 and flows continuously over thedensity gradient in the rotor 12, and viruses sediment out of thestream, banding into the density gradient according to buoyant density.This pumping of sample into and out of the rotor 12 can be performedwith the centrifuge spinning at high speed. The continuous stream allowsa large volume of fluid to flow through the annular space 13, whichpermits virus material to be captured in the gradient, even with smallconcentrations of viruses in the fluid. In ordinary zonal operation (notcontinuous-flow), the sample does not flow continuously into the rotorfor long periods of loading, but rather the entire sample volume, whichmust be less than the annular volume in the rotor, is loaded andenclosed in the rotor 12. The rotor volume is then closed off beforeacceleration to high speed. In either case, this is called the loadingphase of the isopycnic banding separation. After loading andcentrifuging to achieve banding, the virus-containing bands arerecovered by displacing the bands sequentially, with lowest densitybands exiting first and highest density last. As the density of eachvirus uniquely determines the position of that virus or particle in theexiting stream, the timing of the detection of specific virus particlesprovides particle density information.

A fresh gradient is loaded into the rotor 12 by pumping a low densityfluid, containing no cesium chloride, into the rotor 12. As illustratedschematically by the presence of two fluid tanks and a mixing valve inpart 15 of FIG. 1, a high density fluid, typically containing about 60%cesium chloride is mixed with the low density fluid at a variablehigh:low ratio, which via PC control increases with time until theloading is complete. The fluids pass through the fluid entry ports 14 atthe top of the annular space 13. Concurrently, the rotor 12 is spinningat a low speed of about 4,000 rpm, with the speed being controlled bythe timer control system in tandem with the fluid entry anddisplacement.

After the fresh gradient is loaded, the control system actuates valveswhich flow fluid through the rotor 12 in the opposite direction, pumpingsample from the holding tank 6, through switch 10 (in the firstposition), through the bottom entry port 50, and upward through theannular space 13, entering at the bottom end and displacing fluid out atthe top of the rotor 12 through 14 and out discharge port 37. Afterestablishing flow reversal, the control system initiates and regulatesthe centrifuge to a preferred rotational speed of about 60,000 rpm for aB-series rotor. In extremely dry environments, water exiting thecentrifuge may be recycled back into the system by pumping it back intothe collector 1 where it can be used for air scrubbing. At a rotationalrate of 60,000 rpm and flow rate as high as 6 liters/hr, over 90% of allvirus enters the gradient from the sample fluid stream, where it remainsimprisoned. After on the order of 10–30 minutes of operation, whichallows as much as 3 liters of sample fluid to pass through the rotor 12,the inflow and effluent flow are shut off, and the high-speed rotationcontinues for an additional 30 minutes to band the viruses. The virusesbecome banded in the gradient. The centrifuge controls are actuated by atimer-regulated control system, which is preferably a standardPC-computer interface.

In operation, sample liquid is introduced into the density gradientwithin the centrifuge rotor at the low-density end of the gradient, andeach particle or molecule penetrates into the gradient at a rate thatincreases with the mass of the particle, and with the density. In thecase of a protein molecule, the mass is much smaller than that of avirus by at least an order of magnitude, and the density is about thesame as that of a relatively low-density virus. Accordingly, the rate ofbanding for proteins is much slower than for viruses. The centrifugationis run just long enough for the smallest virus particles of interest tohave enough time to band to the desired resolution in the gradient. Thisis typically within about 1–5% of the equilibrium position. The proteinswill then primarily be to the low-density side of their equilibriumpositions, as they started on that side. Since the equilibrium positionof most proteins in a gradient is nominally about 1.3 g/ml, at the endof this shortened operating time, most proteins are positionedconsiderably lower than 1.3. The proteins are at positions which are notcollected, and not sent on to the next stage as they are outside of the“virus window”. Accordingly, the density-gradient centrifugation steptakes on some of the properties of a combined two-stagedensity-gradient/sedimentation coefficient separation.

Once the viruses are banded, the centrifuge is decelerated to low speed,and the gradient is recovered by pumping the dense fluid of preferably60% CsCl from the gradient supply system 15 to the outer edge of theannular space 13 through 14. The dense fluid displaces the gradient,with low density bands exiting first followed by high density bands.After gradient removal, the high density material in the rotor 12 isdisplaced by low density fluid, which enters from the inner rim of theannular space 13 at point 50 and displaces the high density materialfrom the outer edge of the annular space 13. The procedure is completein a few minutes, and the cycle repeats again beginning with the loadingof the density gradient at low speed.

Ultracentrifugation provides the advantages of desorption of virusesfrom fomites and universal capture of all catalogued and non-catalogedviruses, with high capture efficiencies of greater than 95%.Ultracentrifugation also is not dependent on biochemical reagents, andprovides a high degree of virus separation from the backgroundcomponents. Additionally, density information of the viruses is providedby the ultracentrifugation, providing the y-coordinate in the VirusWindow plot, discussed herein. The coaxial seal 11 is commerciallyavailable as a Beckman's Zone Assembly, part no. 334241. Examples of thecentrifuge rotor are the U.S. Army's B-VIII, B-IX and B-XVI, orpreferably the Beckman Spinco CF-32 Ti Continuous Flow Rotor, orBeckman's Z-60 rotor for ordinary zonal centrifugation. For thecentrifuge itself, the Beckman Optima XL-100K PreparativeUltracentrifuge is well-suited for all of these rotors.

The results of the extraction of the ultracentrifugation of thecentrifuge rotor 12 are analyzed from biological background by means ofa “Virus Window”. The Virus Window is a density-size (r-d) ordensity-sedimentation coefficient (r-S) plot of biological componentswhich are pathogenic to humans, with the x-axis showing either size d orsedimentation coefficient S, and the y-axis showing density r, as shownin FIG. 6. Most Mammalian viruses are approximately between 1.175 and1.46 gm/ml density and have a diameter between about 22 and 200nanometers (or, alternatively rephrasing this size range, withsedimentation coefficient between 120 and 6,000 Svedberg units). TheVirus Window for plant viruses will be different. The Virus Window ofFIG. 6 is an extremely useful concept not only because it shows howviruses can be separated from other non-viral background, but alsobecause the different virus families are substantially separable fromeach other. Within the Virus Window, each virus family is distinguishedby a particular rectangle with little overlap between the 20 familyrectangles. Accordingly, with a known density and size, the detectedvirus particle is pinpointed to its particular family in the VirusWindow. In any case, particles with densities and sizes that both fallin the Virus Window ranges can, with high confidence, be presumed to beviruses; thus when counts are registered in the detector of the presentinvention, having previously been selected by centrifugation for densityin the range of about 1.175 to 1.46, and further selected by theDifferential Mobility Analyzer for size between about 22 and 200 nm,then it can be concluded with a high degree of confidence that theseindicate the presence of viruses in the sample. Furthermore, thisconfidence level is further increased if the density and size fall intoa particular region of the Virus Window known to correspond to a virus.Similarly, other particles of potential interest in detection—such asprions, other virus-like particles, and other natural or artificialparticles, colloids, cell structures, or macromolecules—will frequentlyhave unique positions in the density-size plot that may allow them to beseparated from other components and thereby be detected in the presentinvention.

Although to a very large degree only pathogenic viruses fall within theVirus Window, other background components fall close to the VirusWindow. These components are microsomes and similar sub-cellularstructures. These components can be effectively eliminated by addingnonionic surfactant, such as diethylene glycol monohexyl ether, to thecollection stage 101 exit stream at inlet 4. The surfactant solubilizesthe microsomes and membrane fragments. As recovery of viable viruses isnot necessary, release agents can be used. The release agents arepreferably organic solvents and surfactants, more preferablyamphiphiles, and most preferably low molecular weight amphiphiles suchas diethylene glycol monohexyl ether. The release agents provide severaluseful effects. First, they act to break up and even dissolve cellularsubstructures, such as microsomes, ribosomes, lysosomes, peroxisomes,and mitochondria, which have sizes and densities similar to viruses andset the limit on the required resolution, in the case of detection ofviruses. Second, upon dissolution of the lipid envelope with suchagents, the increase in the virus density is significant (the density ofthe viral core, which is the virus minus its lipid envelope, is ingeneral significantly higher than that of the enveloped virus). In thecase of hepadnaviridae, for example, this may be from about 1.25 to1.36. Both effects serve to further differentiate viruses from,particularly, microsomes in the Virus Window plot, the first by actingto eliminate the microsomes, and the second by increasing the differencein density between the viruses and the background microsomes. Third,release agents enhance the desorption of viruses from solid matter,which is particularly important in the detection of airborne viruses.Release agents can also break up aggregates of viruses, especiallyaggregates of encapsulated viruses. The present invention minimizes thisaggregation problem in other ways besides the use of release agents. Thecentrifugation can be performed without pelleting. Consequently, buoyantdensity, and thus isopycnic banding, is not greatly affected byaggregation under these circumstances. (Indeed, banding times arefavorably reduced in the case of aggregation, and techniques can beapplied that take advantage of this, within the broad context of thepresent invention). Any aggregation will generally produce only a smallshift in, and/or broadening of, resulting virus bands. The portion ofthis exiting stream that contains the Virus Window is pumped to thepurification stage 103 with the position of a particle along this streamgiving the density of that particle. The useful part of the stream, inthe case of general virus detection where the range 1.175–1.46 is passedto the next stage, is in the preferred embodiment on the order of about10 ml; thus, this stage does not effect a large increase in virusconcentration, though it does effect a very large increase in theconcentration of viruses relative to other non-viral components.

Although feasible, a separate centrifugation to separate particles bysedimentation coefficient for Virus Window x-coordinate information isnot necessary. A Differential Mobility Analyzer (DMA) 26, which asdescribed below, provides rapid analysis of particle size. Additionally,separation of viruses from soluble proteins can also be done in thepurification stage 103. An even further separation of proteins, andother macromolecules smaller than viruses, from viruses can also be doneby tuning the supersaturation in a condensation particle counter so asto not detect macromolecules as small as proteins. The centrifugedimension and rotor speed for optimal centrifugation can be calculated.Optimal times are preferably thirty minutes or less and resolutions arepreferably 0.02 density units (0.02 gm/ml) or better.

The sample fluid passes from the extraction stage 102 into thepurification stage 103. Typically, this could be in the form of 15pulses, each on the order of 1–10 ml in volume, and each correspondingto a density slice with a width on the order of 0.02 gm/ml. In thepurification stage 103, a membrane filter 22 separates the viruses fromsoluble proteins (removing the need for a second, sedimentation ratecentrifugation in the previous extraction stage 102), and concentratesparticles with sizes greater than the pore size into a very small volumeof liquid; additionally, in this stage soluble salts, including thosefrom the sample as well as the density gradient material (e.g., cesiumchloride), are greatly reduced in concentration. The membrane filter 22may be Millipore's VIRESOLVE Membrane, an AMICON P membrane, orpreferably a Pall FILTRON OMEGA Series membrane with a 1,000,000molecular weight cutoff. The water permeability of the membrane filter22 is on the order of 0.01 ml/cm2-sec-psi, so that a membrane area of0.1 cm2 yields a flux of order 6 ml/min at 100 psig transmembranepressure. The membrane filter 22 is incorporated into a housing which isdesigned to allow flow rates on the order of 0.1–20 ml/min duringfiltration, which results in loading of the filter with particles largerthan about 15 nm (which includes all virus particles), after which theparticles are confined within a small front-face-side collection volume.A small-volume filtration filter holder 21, such as Schleicher &Schuell's SELECTRON, is used to hold the membrane filter 22. Morepreferably, a filter holder with a design like that of the SELECTRON,but made out of an alternative material which does not degradeelectrolytically under high voltage, is used.

A four-way positioner 30 in the purification stage 103 allows automatedprocessing of particles in the membrane filter 22. The positioner 30 isdriven by a computer-controlled motor which positions the filter holderin one of four ports.

In the first position, the positioner 30 positions the membrane filter22 to accept the sample flow outputted from the extraction stage 102.Each 0.02 gm/ml density slice from the output of the extraction stage102 is, after passing through switch 10 in the second position, loadedthrough the membrane filter 22 in less than about 2 minutes;alternatively, larger density slices can be filtered, requiringappropriately longer times. A standard 0.2 micron poresize filter (suchas available from Corning Costar) is preferably incorporated in theconnection between the output from 102 and the input to 103, in order toremove any remaining particles greater than about 200 nm in size.

When the positioner 30 is switched to the second position, a valvecloses off the sample flow and CsCl-free water from pump 18 out of tank17 which has an inlet 16 is passed across the membrane filter 22 usingon the order of 5 ml of water with a flux time of order 1 minute. Thisreduces the 30% CsCl aqueous solution surrounding the particles to lessthan 100 ppm CsCl, and allows recovery of the CsCl for recycling.Additionally, the amphiphile, viscosity additives and buffer componentsare reduced in the membrane filter 22. More preferably, ammonium acetatesolution, with on the order of 20 mM concentration in water, is used forthis operation, preparing the liquid for downstream detector stageoperation.

On switching the positioner 30 to the third position 19, the pure water(or ammonium acetate solution) is shut off, and a final filtration isperformed in order to reduce the volume of liquid on the retentate sideof the membrane, thereby greatly increasing the concentration of virusesand reducing the volume of liquid to the small quantities required foroperation of the detection stage 104; the filtrate in this step passesout through port 23. More precisely, the purification stage 103 isintegrated with the electrospray assembly 24 of the detection stage 104by a punctured disk fitting. The fitting has a 150 micron hole drilledthrough a tubular stub in its center. When positioner 30 is in the thirdposition, this hole allows the filtrate to pass out through port 23.When the positioner 30 is in the fourth position, the inlet end of theelectrospray capillary 29 (the end opposite the spray tip) is insertedinto this 150 micron hole. This fits in a piston-like manner into thestainless steel cylinder of the SELECTRON (or SELECTRON-like) filterholder. The cylinder slides over the steel disk, and is positioned witha gap between the steel disk and the ultrafilter surface on the order of100 microns.

In the fourth position 20, in accordance with the above, the membranefilter 22 is positioned for entry of the virus containing retentate intoan electrospray capillary 29 of the detection stage 104. (Alternatively,instead of fluid passing directly from the purification stage 103 to theelectrospray, an intermediate component may be used to accomplish afurther purification and/or concentration). A platinum wire may be runfrom the voltage source of the electrospray unit 24 to the interior ofthe liquid inside the volume on the retentate side of the membranefilter, in order to establish a current return for the electrosprayoperation.

The detection stage 104 accomplishes several functions, which include afinal purification, an individual virus particle count and a sizedetermination of the detected particles. The detection stage 104includes three major components, an electrospray assembly (ES) 24, aDifferential Mobility Analyzer (DMA) 26 and a Condensation NucleusCounter (CNC) 27, which is alternatively called a Condensation ParticleCounter (CPC). The components may be commercially obtained individuallyfrom TSI, Inc. of St. Paul, Minn. The Condensation Nucleus Counter 27and Differential Mobility Analyzer 26 units are also availablecommercially from TSI as a single integrated unit, which can beaccompanied by an IBM PC with associated software. This allows for aninexpensive set up compared to a mass spectrometer. The detection stage104 can conduct measurements concurrently with the collector 1 obtainingthe next cycle's collection.

Passing from the purification stage 103, the retentate enters thedetection stage 104 at the inlet of the electrospray capillary 29 of theelectrospray assembly 24 in the fourth position of the positioner 30.Entry into the electrospray capillary 29 is done without passing theretentate through piping, which might cause sample losses. Theelectrospray capillary 29 is on the order of 25 cm in length, and theinlet of the electrospray capillary 29 is positioned to the smallfront-face-side collection volume of the UF membrane 22, as describedabove. The electrospray capillary 29 is then positioned to sample liquidfrom the retentate-side of the filter and the sample liquid enters theelectrospray assembly 24.

In the electrospray assembly 24, the liquid sample solution is passedinto an orifice or “jet” of 50 micron diameter, and droplets are ejectedunder the influence of an electric field. The droplets are typicallybetween 0.1 and 0.3 microns in size, with a fairly narrow sizedistribution. At a droplet size of 0.3 micron, sampling rates are 50nl/min (50 nanoliters/minute), allowing the electrospray assembly 24 tospray the collection volume in on the order of 20 minutes permicroliter.

From the electrospray assembly 24, the sample passes to a chargeneutralizer 25. The charge on the droplets is then rapidly recoveredusing an ionizing atmosphere to prevent Rayleigh disintegration. Theneutralized ES droplets are then dried in flight, leaving the targetvirus molecules and/or dried residue of soluble impurities. From thecharge neutralizer 25, the target virus molecules and/or dried residueenters the Differential Mobility Analyzer 26.

The Differential Mobility Analyzer 26 uses electrophoretic mobility ofaerosol particles to classify the particles by size, using the inverserelationship between the mobility of a particle to its size. In theDifferential Mobility Analyzer 26, particles are carried by an airstream at a set velocity through an electric field created by a chargedrod. If the particle is singly and positively charged, it experiences anelectrostatic attraction to the rod, which competes with the inertialforce of the flow. When the electrophoretic mobility falls in a certainrange, the particles pass through a narrow exit port at the end of thecharged rod. The particle size range, which is generally 0.01 to 1micron, is divided into 147 size channels. The entire range isautomatically scanned in 1 to 10 minutes, generally 3 minutes. TheDifferential Mobility Analyzer 26 has only a possible 3% instrumentalerror for virus size determination. Additionally, there is a possiblesize increase due to the covering of the virus particle with impurityresidue, which at an impurity level of 100 ppm, a typical 40 nm virushas a possible error of up to about 2% in effective size. If theimpurity levels are less than 20 ppm, the error becomes smaller than 1%.

When the primary droplets from the electrospray assembly 24 are 0.3micron, a 1 ppm soluble impurity creates a 3 nm residue particle, and a125 ppm soluble impurity creates a 15 nm particle. Particles which are15 nm in diameter can be separated in the Differential Mobility Analyzer26 from viruses which are at least 22 nm in diameter. Accordingly,soluble impurities must be reduced to less than 100 ppm (0.01%) to avoidbackground interference with virus signals.

Detection of proteins at levels of 10¹¹–10¹² molecules/ml indicates thata sensitivity level for viruses of 10¹⁰ particles/ml can be achieved,and possibly 10⁹ particles/ml, particularly by combining theDifferential Mobility Analyzer 26 selection with an adjustment of theKelvin radius of approximately 10 nm. Impurities of 1 ppm yields a 3 nmresidue particle which can overlap protein sizes. Impurity levels of 100ppm or less are acceptable in the detection of viruses, since virusesare several times larger than proteins. Sensitivities of 10¹⁰molecules/ml and possibly 10⁹ molecules/ml are projected based ondocumented results using proteins. In one of the Examples given below,detection of 10¹² pfu/ml (a pfu is a plaque-forming unit) was easilyaccomplished even after dilution by a factor of 128, demonstratingdetection at a level of 10¹⁰ pfu/ml.

The Differential Mobility Analyzer 26 validates against false positivesby changing the dilution and seeing whether the particle size alsochanges. Additionally, the Differential Mobility Analyzer 26 can be usedto provide another layer of protection against interference fromimpurities up to the 100 ppm level. The level of 10¹⁰ molecules/mlcorresponds to 2×10⁷ viruses in a 2 microliter collection volume of thepurification stage 103, and 10⁹ molecules/ml corresponds to 2×10⁶viruses. At a collection volume of 10⁷ viruses of the present invention,or minutes of XM2 sampling, 20,000 liters (20 m3) of air are sampled.Accordingly, the sensitivity of the present invention is on the order of500 viruses per liter of air. With impurity levels of 100 ppm or less,virus size can be determined by the Differential Mobility Analyzer 26 towithin about 4%. The detection stage requires on the order of 5 to 40minutes, including Differential Mobility Analyzer 26 size determination,and can be preformed concurrently with centrifugation for a subsequentcycle.

From the Differential Mobility Analyzer 26, the sample enters theCondensation Nucleus Counter 27, which uses a nucleation effect. Theaerosol sample enters and passes through a heated conduit having anatmosphere which is saturated in butanol. The sample is routed into acooled condenser, where butanol vapor condenses onto the sampleparticles, which act as nuclei. The saturation is regulated so that nocondensation occurs on the nuclei below a critical size, which limitsfalse background counts to less than 0.01 particle/ml. With nucleatingparticles, condensed droplets grow to micron size and are opticallydetected using a 780 nm laser diode with photodetector. Provided thatthe level of impurities is low enough that the residue particles arebelow the threshold of detection by the Condensation Nucleus Counter 27,and/or are separated from the target molecules by size, then only thetarget molecules will be registered with the Condensation NucleusCounter 27. As the nucleation of droplets does not depend on surfacecharacteristics of the particles, butanol saturation can be adjusted fora critical size of 0.01 micron radius which minimizes background countsfrom proteins and other soluble impurities. Response times for stepchanges in concentration are less than 20 seconds, and operation of allcomponents is in the temperature range from 10° C. to 38° C.Supersaturation tuning for a 10 nm Kelvin radius threshold in theCondensation Nucleus Counter 27 can be used to cancel the detection ofnon-viral impurities, including proteins, provided they are below about100 ppm.

The purification stage has an output volumetric rate which is very wellsuited for input into the ES-DMA-CNC particle counter, which addressesthe strict requirements and narrow range of operating parameters for theES-DMA-CNC unit. In recognizing the high value of this molecule-countingand molecule-sizing ES-DMA-CNC unit, filtration provides excellentsamples for the purification/concentration stage prior to this detector.The ES-DMA-CNC combination allows particles to be sized and permitsimproved sensitivity by an order of magnitude over a DMA-CNCcombination. Protein concentrations of 10 mg/ml, or 10¹¹–10¹²molecules/ml, can be detected and sized.

The system is controlled by a computer 28. When data collection andinstrument control are handled by the same computer, the computer mayvary the mode of operation in response to virus detection. Initially,before viruses have been detected, the system places the entire 300 mlof density gradient from the extraction stage 102 through the membranefilter 22 to scan all virus sizes from 22 to 200 nm. Alternatively, theDifferential Mobility Analyzer 26 is by-passed entirely, provided thatnon-viral concentrations are low enough that tuning of the Kelvin radiusin the Condensation Nucleus Counter 27 is sufficient to reducebackground. Once viruses are detected, the Differential MobilityAnalyzer 26 indicates the sizes of the viruses detected. The computercan then trigger the output of the extraction stage 102 to be sampledpiecewise in the purification stage 103. By breaking the range of virusdensities, which is about 0.3 gm/ml into 10 or 15 slices, the density ofthe detected virus is within about 0.02–0.03 gm/ml, which is sufficientto narrow most viruses down to a single family. Following this, theregion in the centrifuge output stream surrounding this density can bedivided still finer, to provide better accuracy on the viral density.Through data base comparison, the system identifies the viral familiesfrom the measured densities and sizes, and provides output of detectedviruses by density, size, concentration, apparent changes inconcentration over time, and if desired, audible and/or visual alarms inthe presence of detected viruses. Being automated, the instant inventioncan run continuously for long periods of time without an operator. Inaddition to making continuous virus monitoring possible at a largenumber of sites simultaneously without the need for scores ofvirologists, the automation afforded by the present invention alsolimits the risks of viral infection of technicians.

Other potential physical means of separating viruses and other particlesfrom background and/or enriching their concentration may involvecapillary electrophoresis (purification and concentration enrichment),sedimentation-rate centrifugation (primarily purification),hydroextraction (mainly concentration), dialysis (purification andconcentration), organic/inorganic flocculation (purification andconcentration), and capillary chromatography, which can size-exclusion,hydrophobic interaction, or ion-exchange chromatography (purificationand concentration).

EXAMPLES

Analysis of two blind samples was performed, using the filter membranestage and the electrospray (ES)—differential mobility analyzer(DMA)—condensation particle counter (CPC) triage, or gas-phaseelectrophoretic mobility molecular analyzer (GEMMA).

Filtration: Two samples, labeled as AFO001 and AFO682 were obtained.AFO682 had been collected and contained viruses; AFO001 was a blank orcontrol, although the foregoing was not known about the two samplesprior to testing. Each original sample was on the order of 1.2 ml involume. From each sample, all but about 200 microliters was taken andprefiltered, through a 0.2 micron poresize Millipore syringe filter withlow dead volume. Approximately 400 microliters of this was removed ineach case and processed in a filtration unit designed and built for thispurpose. A 500,000 MW cutoff membrane was selected to separate viruses,which were retained, and to pass proteins and soluble salts out in thefiltrate, which was discarded. Successive diafiltration was used, witheach filtration step concentrating retained material into a volume ofabout 5 microliters on the retentate side of the membrane. Betweensuccessive filtrations, 20 mM ammonium acetate solution was used torestore the volume to about 400 microliters. This strength of ammoniumacetate was used for the proper operation of the electrospray (ES) inthe detection stage. After two successive diafiltration steps, two 100microliter portions of the final, filtered sample were collected in twoways. The first 100 microliters was obtained by forcing out 100microliters of the retentate volume back through a port which wasforward or upstream of the membrane surface, during the final leg of thelast diafiltration. This was done after allowing 20 minutes fordiffusion of the viruses away from the membrane surface. The second 100microliters was obtained by using a gas-tight syringe press-fit into thefiltrate outlet port, to push ammonium acetate buffer backward acrossthe membrane, from the filtrate side to the retentate side, in order toelute virus from the membrane. The design of the filtration unit wassuch that the retentate side of the membrane remained in a waterenvironment, avoiding for example, an air-purge or vacuum flush of thesystem causing irreversible adsorption and breaking of virions.

Detection: The filtration resulted in three 200 microliter samples foreach of the two specimens: one before filtering, one prefiltered, andone prefiltered and then ultrafiltered. The samples were placed inEppendorf vials. Each sample contained ammonium acetate, which isimportant in maintaining the conductivity necessary for electrosprayoperation. Being volatile, it decomposes and evaporates into ammonia andacetic acid during the in-flight drying of the electrospray-generatedaerosol, and so does not contribute to the final scan. Additionally,each sample contained the viral particles and fragments of interest.Furthermore, each sample contained soluble salts, which are not volatileand thus lead to a “residue particle” after in-flight drying. Forexample, with the electrospray set at 300 nm droplets, a salt level of 1ppm would yield an average residue droplet diameter of (1/10⁶)^(1/3)×300nm=3 nm. Very high salt concentrations can increase conductivity anddestabilize the electrospray, which in all the data shown, was not aproblem. However, in some scans shown for samples that were notfiltered, the salt peak from these residue particles extends out to ashigh as 15 nm.

Samples were input by inserting a capillary into the Eppendorf vial.Flushes of pure, 20 mM ammonium acetate were run before and betweensamples. Stability of the electrospray is in general indicated by acurrent between 200 and 400 nA.

Results: FIGS. 2A and 2B each compare DMA-CNC data for a typical samplebefore and after ultrafiltration, both being pre-filtered. In each ofthese two figures, the solid curve shows data before ultrafiltering andthe dashed curve after ultrafiltering. In FIG. 2B the peaks centered atabout 9.0 nanometers and at about 4.2 nanometers represent backgroundpeaks due to soluble impurities (mainly salts) before and afterultrafiltration, respectively.

FIG. 2A, which focuses on the 15–40 nm range where the number of countsis low, show a solid curve for the ‘before ultrafiltered’, which wasdiluted by a factor of 16 before running, with a dashed curve for theultrafiltered sample run at full strength. This plot, which wasreproduced over a number of scans, shows the two curves tracking eachother well into the region above 15 nm, with the ratio starting wellabove 1:16 (roughly 1:4) but approaching this at larger diameters. Thisshows that the ultrafiltration retained this material well, at least asthe diameter increased to above 20 nm, but even fairly well between 15and 20 nm; the MW cut-off curve of the membrane filter rises from 0 to90+% retention between about 10 and 20 nm., significant since intactvirus particles, those lying in families known to be infectious tomammals, are always greater than 20 nm.

In FIG. 2B, the dashed curve shows data after ultrafiltration incomparison with the solid curve before ultrafiltration. FIG. 2B shows agreatly reduced salt content, given by the cube of the diameter ratio,and remarkable reductions in protein concentrations. The proteinconcentration are in the range of 7–15 nm, in which many proteins lie,particularly, proteins higher than about 80,000 MW. The data under thesolid curve are for the non-ultrafiltered sample at 1/16th strength, sothat the reduction is even more dramatic than as appears in the plot. Asplot 2A, above, showed that the 15+nm fraction was preserved by theultrafiltration, even quantitatively above 20 nm. The reduction in thesalt peak was also extremely good, both in intensity and in the lowereddiameter, pushing it down away from the region of interest. Thisindicates that the ultrafiltration methodology is extremely effectivefor removing soluble salts and proteins, while preserving 20 nm andgreater fractions. It is evident that the ultrafiltration reduced thebackground, which is due to the salt residue particles, both inmagnitude and location, shifting the peak position for the backgroundfrom about 11 nm to about 5 nm, putting it well below the range ofinterest for virus detection.

The experimental results of an environmental source sample was analyzedwith the filter-ES-DMA-CNC combination. As a blinded experiment, thecomponents of the two samples were not known, except that one samplecould be a “blank”, with no viruses, and the other sample was eitherdoped with virus or was an environmental sample, collected from samplingof air in the wild. Upon analysis of the DMA-CNC data, viruses weredetected and counted in one sample, AFO682, and no viruses detected inthe second sample.

FIG. 3 shows a plot of the GEMMA data for the two ultrafiltered samples.The x-axis gives the particle size in nanometers, and the Y-axis gives aconcentration measurement. Evidence of particles in the range of 22–40nm was shown, the range for intact virus particles, in sample AFO682. Insample 682, the counts here are quite low, and translated intoconcentrations on the order of 10¹⁰ particles/ml or 10 femtomoles perml, after filtering. In sample AFO001, there is essentially no activitymeasured in the range 22–40 nm, where background counts are typically 0,or at most 8 counts. With isopycnic banding information from thecentrifuge stage, a double-check to distinguish between simple proteinaggregates or polysaccharides with gravimetric densities of about 1.3 orless, and unenveloped viruses of about 1.4 is possible. Upon use of thefull system including centrifuge, the locations of the viral families indensity-size space could be mapped systematically, providing a look-uptable that would be useful for distinguishing between viral andnon-viral material.

FIG. 4 shows data for a similar analysis on a known sample, prepared ofMS2 bacteriophage of known concentration, 10¹² pfu/ml. Afterultrafiltering 0.5 ml of the known sample as described above for theblind samples, without prefiltering, a 50 microliter sample was analyzedwith the ES-DMA-CNC combination. FIG. 4 shows that the virus was easilydetected, and the virions are counted and sized. The size obtained fromthe DMA was 26 nm, in agreement with literature for scanning electronmicroscopy (SEM) analysis on Leviviridae. The linewidth (full-width athalf-max, or FWHM) was small at only 2 nm, indicating that the size canbe determined very accurately and that viruses of a single type can bedistinguished from other viruses and non-viral particles with highreliability.

In addition, the ultrafiltered known sample in FIG. 4 was dilutedsuccessively by factors of 2, down to a dilution of 128, and analyzed inthe ES-DMA-CNC. Even at 1/128, the peak was still easily distinguished,giving a signal-to-noise ratio of approximately 10:1. FIG. 5, shows thepeak amplitudes and areas which are plotted as a function of dilution(relative sample concentration, with the undiluted sample having a valueof unity). FIG. 5 demonstrates the linearity of the detection method.

FIG. 8 discloses one embodiment of an arrangement 110 of utilizingconcentration, purification and detection devices for detecting thepresence of virus particles. The detection apparatus 110 includes aninput control section 111 for receiving a test sample through inlet 112and two gas-phase electrophoretic mobility molecular analyzers (GEMMA)126 and 156, each of which comprises the electrospray (ES), thedifferential mobility analyzer (DMA), and the condensation particlecounter (CPC) assembly, as described above. The arrangement of FIG. 8also includes ultrafilter modules 114 and 135 which are selectively usedwith the GEMMAs 126 and 156 for detecting the presence of virusparticles in various types of samples. In a first configuration, theapparatus can be configured to process a “dirty sample”, which isdefined as a sample containing a known virus of along with otherimpurities, such as growth media, salts and proteins. The dirty sampleis fed through conduit or tube 113 to a first ultra-filtration(UF)module 114, as shown in FIGS. 9 and 10, where the sample is furtherconcentrated. The first ultrafiltration (UF) module 114 could utilize across-flow type of ultrafilter 116, as depicted in cross-section in FIG.10, where the smaller size or smaller molecular weight particles flowoutwardly through the walls of the filter 116 while the larger virusparticles are retained therein. Sequential arrangements of differentpore size ultrafilters can be picked to selectively control the flow ofa particle with a chosen size range so that the chosen particles canflow through the walls of a first filter, such as a cross flow filter,and then not pass through the walls of second filter to thereby purifyand concentrate a fluid sample limited to particles within the chosensize range. The sample retained within the filter is then fed to theGEMMA unit 126 to determine the concentration of the virus particles. Ifthe test results from the GEMMA unit 126 suggest that the concentrationof the virus is too dilute, then the sample can be fed to a secondultra-filtration (UF) module 135, where the sample can be furtherpurified and concentrated. When desired, an ultracentrifuge 108 can beselectively coupled either to the input control section 111 or anultrafiltration module 114 after the samples have been separated intogradient density bands.

A second configuration is shown in FIG. 8 where the apparatus isconfigured to process a “clean sample”, which is defined as a samplecontaining a known virus with few impurities so that the sample can befed directly to a GEMMA unit 126. If the results from the GEMMA unit 126suggest that the sample is too dilute, then the sample can be furtherconcentrated by feeding the sample through conduit 129 to the secondultra-filtration module 135. To coordinate and calibrate the resultsfrom the GEMMA, a calibration means or tracer solution or biomarkermeans 145 of known concentration, such as MS2 bacteriophage, can beintroduced into the conduits 113, 125, and 131, as represented by itemnumber 104, so that the concentrations results indicated by the GEMMA'scan be compared and calibrated. Where a known virus “test” sample isused, the test or calibration sample can be fed through conduit 125directly to the first GEMMA unit 126. If the test results do not showthe presence of the particular known virus, then the sample is thenfurther concentrated in the second ultra-filtration module 135.

A third configuration is also shown in FIG. 8 where a sample is “clean”but may be of dilute concentration. For this configuration, the sampleis fed directly to the second ultrafiltration module 135 for furtherconcentration. The concentrated sample is then fed to a second GEMMAunit 156.

With an ultrafiltration module 114, such as generally depicted in FIG.9, a sample is first placed in the feed reservoir 117 and then theperistaltic pump 118 is turned on to cause the sample to flow throughthe filter container 115. As the sample is fed through the tubular orcross-flow filter 116 housed within the filter container, the filtrate,which may include salts and proteins, is forced through the filter 116leaving the viruses in the sample contained within the filter, asrepresented in FIG. 10. The speed and pressure of the peristaltic pump118 and the settings of the inlet-outlet valve 121 can be adjusted tocontrol the internal pressure within the system, as monitored by thepressure gauges 119 and 120. A first ultra-filtration module can beused, for example, to reduce the sample volume from about 5 millilitersto about 500 micro-liters.

FIGS. 11A and 11B illustrate different arrangements of the virusdetection system of FIG. 8. In FIG. 11A, the system comprises acollecting means 160 which may include the collecting means 101,ultracentrifuge 102, a fluid container, or some other form of liquidcontainer for a sample. The output of the collecting means 160 thenpasses to a purifying and concentrating means 165, which may be a filterassembly as shown in FIGS. 9 and 10, for cleansing the liquid sample andfor concentrating a particular size range of particles. The output ofthe purifying and concentrating means 165 is then fed to detecting means156, in the form of the GEMMA unit. FIG. 11B illustrates anotherparticular arrangement of the virus collection system where acalibration or tracer material 138 of known concentration and size, suchas MS2 bacteriophage that is essentially a biomarker, is inserted intothe collected sample and where a valve means 166 is connected to anoptical measurement means and a computer for controlling the flow pathof the sample.

Adding a biomarker, such as the bacteriophage MS-2, or other calibrationor tracer material to the system provides a means for verification ofinstrument operation and a means for the calibration of other, similarsubmicron size particles. Other viruses and submicron size particleswill have a correlation to their actual concentration in a sample. Thebiomarker or calibration material can be added early in the collectionprocess in a known quantity, of for example one milliliter, and knownconcentration. This one militer of liquid is then run through the GEMMAcounter to determine a count, of for example 1000 bacteriophage. Whenthis biomarker or calibration material is then added to an unknownsample volume it provides a ready reference, since when the unknownsample is reduced to a one milliliter sample volume, it would beexpected to give a bacteriophage count of about 1000. Thus, use of abiomarker or other tracer material provides an accurate method forcalibration of the counting, concentration and purification means. Theuse of a biomarker or tracer material throughout the system also allowsthe system to be adjusted and calibrated.

Valve means 166 may include a turbidity or an optical density measuringdevice or some other device for measuring the “cleanliness” of thecollected sample. Use of a optical or turbidity meter normallyencompasses the shining of a light beam into a fluid sample. A photocellor other light sensitive means measures, for example, the intensity ofthe light that is transmitted through the liquid sample. Clear water,for eample, can be calibrated to be at the 100% level and as the waterbecomes more clouded with other material the percentage transmission oflight diminishes. A light meter means can be connected to a valve means166, a computer means 168 or other control means. For example, the valvemeans can be used to determine if the sample can be fed directly to theGEMMA 126 or if the sample needs to be filtered in the purifying andconcentrating means 114 before being sent to the GEMMA 126. A computermeans 168 can be connected to the valve means 166, the GEMMA 126, andthe second GEMMA 156 for controlling the flow of the sample and fordetecting the concentration of the submicron size particles in thesample.

A. Tests in Removing Complex Media From MS2 Bacteriophage Cultures

A.1. Background

To demonstrate the applicability of the apparatus for detecting virusesin samples, tests were made for removing complex growth media and otherimpurities, such as salts, proteins and other material, from the MS2bacteriophage. The MS2 bacteriophage simulates the size characteristicsof viruses.

A sample of MS2 bacteriophage was received from the Life SciencesDivision at Dugway Proving Ground (DPG). This sample was 500 ml of asgrown MS2 bacteriophage, complete with growth media, at a virusconcentration of 1.4×10¹² pfu/ml. The growth media was comprised of L-Bbroth, 10 g Tryptone, 10 parts NaCl and 5 parts yeast extract. The MS2solution was a dark yellow color and is clear. The sample was from Lot#98251.

The MS2 sample was analyzed using the ultra-filtration modules and theGas-phase Electrophoretic Mobility Molecular Analyzer (GEMMA) detector.As noted above, the GEMMA detector consists of an electrospray unit toinject samples into the detector, a differential mobility analyzer and acondensate particle counter.

Several solutions were prepared to explore the ability of theultrafiltration apparatus to remove contaminates and retain viruses ofinterest in solution. One sample solution of albumin, from chicken egg,was prepared at a concentration of 0.02%, by weight, in an ammoniumacetate (0.02M) buffer. To this solution was added MS2 bacteriophage toa concentration of 3×10¹¹ pfu/ml. Another was prepared containing 2.5%cesium chloride (CsCl), by weight, also in the ammonium acetate buffer.To this solution was added MS2 bacteriophage to a concentration of5×10¹¹ pfu/ml. The MS2 bacteriophage, in both cases, was a highlypurified sample obtained from DPG Life Sciences Division (Lot #98251).

A.2. Results of MS2 plus Growth Media

The mixed MS2 sample, with 1.4×10¹² pfu/ml was analyzed using the GEMMAvirus detector. The sample was placed neat into the GEMMA analyzer andthe results are shown in FIG. 12. The growth media, with the MS2bacteriophage in solution, produces a graph that displays a very broad,nondescript peak across the area of interest of 24–26 nm. The size rangeof 24–26 nm is the expected size for a MS2 bacteriophage. It is notreadily apparent from the as received sample analysis if the sampleactually contains MS2 in solution. The solution required removal of thegrowth media before any meaningful results could be obtained.

The virus plus growth media sample was purified and concentrated usingan ultrafiltration (UF) process. The UF module, shown in FIGS. 9 and 10were used for processing and retaining a virus species for furtherstudy. The UF stage is a hollow fiber-based tangential or cross flowfiltration system. These filtration systems operate by pumping the feedstream through the hollow fiber, as shown in FIGS. 9 and 10. As thesolution passes through the fiber, the sweeping action of the flow helpsto prevent clogging of the fiber. A pressure differential forces thefiltrate through the fiber, while the virus feed stream is purified andconcentrated. There are available a wide range of pore sizes for thefibers. This filtration technique can reduce volumes from over 5 ml toabout 0.2 ml.

The sample of the DPG MS2 with growth media was processed through theultrafiltration apparatus using the parameters for ultrafiltrationlisted in Table 1.

TABLE 1 UF Parameters for MS2 in Growth Media Sample volume-initial 3 mlPump speed 2 Transducer pressure 15 psig Total buffer wash volume 50 mlSample volume-final 2 ml MWCO of module 500K

By continually washing the sample volume with ammonium acetate buffer(the working fluid of the GEMMA analyzer), the UF apparatus allows theremoval of ions, proteins and all other material that is smaller thanthe 500K molecular weight cut-off (MWCO) of the cross flow filter. TheMS2 bacteriophage is retained in the circulating solution and continuedto be purified by the process. As the 500K MWCO filter will effectivelyretain the MS2, the total wash volume can be significantly larger thanthe initial sample volume. The ultrafiltration of this sample wascompleted in less than 10 minutes. The results of the GEMMA analysis ofthe concentrated and purified sample are shown Table 2, below, and inFIG. 13.

TABLE 2 GEMMA Counts for MS2 Bacteriophage Channel Midpoint ChannelMidpoint Diameter (nm) Counts Diameter (nm) Counts 10.5545 128.2 32.196817.5 10.9411 105.7 33.3762 8.5 11.3419 97.7 34.5989 10.5 11.7574 64.335.8664 7.5 12.1881 37.3 37.1803 6.5 12.6346 50.8 38.5423 2.5 13.097534.2 39.9542 3 13.5773 36.8 41.4178 6.8 14.0746 39.5 42.9351 2.2 14.590234.6 44.5079 5 15.1247 28 46.1384 2 15.6788 41.5 47.8286 1 16.2531 102.149.5807 2 16.8485 110.8 51.397 1 17.4658 80.3 53.2798 0 18.1056 120.755.2316 1 18.7688 129.4 57.2549 0 19.4564 167 59.3523 0 20.1691 175.261.5265 1 20.908 168.6 63.7804 1 21.6739 192.2 66.1169 0 22.4679 973.168.539 1 23.291 5228.2 71.0497 1 24.1442 4429.7 73.6525 1 25.0287 639.976.3506 0 25.9455 73.6 79.1476 1 26.896 31.4 82.047 0 27.8813 25.685.0526 0 28.9026 22 88.1683 2 29.9614 19.5 91.3982 0 31.059 16.2

A.3. Results of MS2 plus Albumin

The sample of 0.02% albumin in ammonium acetate, with the addition of3×10¹¹ pfu/ml of MS2 bacteriophage, was analyzed neat in the GEMMA virusdetector. The MS2 peak is centered around 24 nm. The albumin in thesample is displayed as a very broad peak starting below 10 nm andextending to 20 nm, as shown in FIG. 14.

The sample of albumin plus MS2 was then processed through theultrafiltration apparatus. The parameters for the ultrafiltration areshown in Table 3.

TABLE 3 UF Parameters for Albumin plus MS2 Sample volume-initial 1 mlPump speed 2 Transducer pressure 15 psig Total buffer wash volume 40 mlSample volume-final 0.4 ml MWCO of module 500K

After processing in the ultrafiltration apparatus, the sample wasexamined in the GEMMA virus detector. As shown in FIG. 15, the only peakin evidence is centered on 24 nm. The large peak between 10 and 20 nmwas completely removed. The processing of the sample through theultrafiltration apparatus completely removed the albumin protein, whilethe MS2 bacteriophage was retained.

A.4. Results of MS2 plus Cesium Chloride

The sample of 2.5% CsCl, by weight, in ammonium acetate, with theaddition of 5×10¹¹ pfu/ml of MS2 bacteriophage, was analyzed neat in theGEMMA virus detector. As shown in FIG. 16, the MS2 peak is centeredaround 24 nm. The CsCl in the sample is displayed as a very broad peakstarting below 10 nm and extending to over 20 nm. Any higherconcentrations of CsCl would start to obscure the MS2 peak position.

The sample of CsCl plus MS2 was then processed through theultrafiltration apparatus. The parameters for the ultrafiltration areshown in Table 4.

TABLE 4 UF Parameters for CsCl plus MS2 Sample volume-initial 1 ml Pumpspeed 2 Transducer pressure 15 psig Total buffer wash volume 30 mlSample volume-final 0.5 ml MWCO of module 500K

After processing in the ultrafiltration apparatus, the sample wasexamined in the GEMMA virus detector. As shown in FIG. 17, the MS2 peakis shown centered on 24 nm. The large peak between 10 and 22 nm wassignificantly removed. There was a small remnant of the CsCl peak in theprocessed sample due to the smaller amount of buffer wash volume in thiscycle. To completely remove the CsCl, the ultrafiltration process wouldonly need to be continued with further washing until all of the salt wasreplaced with buffer solution. The processing of this sample through theultrafiltration apparatus also retained the MS2 bacteriophage.

A.5. Analysis

The ultrafiltration apparatus was very effective in removing the growthmedia from the solution of MS2 bacteriophage. The addition ofapproximately ten times the amount of starting solution with ammoniumacetate buffer (3 ml vs. 50 ml respectively) allowed the efficientreplacement of the growth media with the buffer solution. The backgroundof the GEMMA scan of the ultrafiltration-processed solution was very lowdue to the low detection of ammonium acetate. In addition, theultrafiltration process for comparable volumes can be completed inapproximately 10 minutes.

The addition of other contaminating materials in a virus solution canalso be successfully removed from solution while retaining the virus.The albumin protein was almost completely removed from the MS2containing solution by ultrafiltration. The adjustment (if necessary) ofthe pore size of the ultrafiltration modules allows for greatflexibility in the processing of solutions.

The CsCl solution appeared to require further washing to completelyremove the salt from the virus containing solution. From the tests todate, it appears that the wash volume for the removal of CsCl in theultrafiltration apparatus requires the initial sample volume to bewashed with approximately 40–50 times the volume of buffer solution, forcertain impurities, to completely remove those impurities.

B. Tests of Effective Filter Size in Concentrating MS2 Bacteriophages

Nominal molecular weight cut off values (MWCO) of various filters hasoften lead to the assumption that items larger than the cut off valueswill be retained after filtration. It was discovered that, at least forMS2 bacteriophage, there are exceptions. It was discovered during thefiltration operation, that counts of MS2 decreased during repeatedcycles of ultrafiltration and purification. This was an importantdiscovery in that for the detection of small numbers of viruses, anyloss may be important. As a result, this study was initiated to betterunderstand the cross-flow filtration characteristics of MS2bacteriophage. The sample of MS2 bacteriophage, used in the filtrationstudies, was received from the Life Sciences Division at Dugway ProvingGround (DPG). This sample was 2 ml of purified MS2 bacteriophage at aconcentration 1×10¹⁴ pfu/ml or 10.2 mg protein/ml. This highly purifiedsample is from Lot #98110.

The two types of filters used in this study were a centrifuge tubeassembly, where the solution is forced through the filter bygravitational forces and a cross flow filter apparatus of FIGS. 9 and 10with pressure pushing the solution through the filter. The centrifugefilter assemblies are available in various sizes and molecular weightcut off (MWCO) filter inserts. The MWCO is changed to capture biologicalmaterial, such as proteins, cell products and viruses, by molecularweight differentiation. The cross flow filter, or ultrafiltrationapparatus, is also used to capture or reject biological material byadjusting the MWCO of the filter. These filtration systems operate bypumping the feed stream through a hollow fiber. As the solution passesthrough the fiber, the sweeping action of the flow helps to preventclogging of the fiber. A pressure differential forces the filtratethrough the fiber, while the biological feed stream is purified andconcentrated. There are available a wide range of pore sizes for thecentrifuge filters as well as the hollow fiber filters.

The MS2 samples were analyzed after filtration using the GEMMA detector,consisting of an Electrospray unit to inject samples into the detector,a Differential Mobility Analyzer and a Condensate Particle Counter.

B.1 Test Solutions

The first set of solutions,consisted of 1×10¹¹ pfu/ml of MS2 in a cesiumchloride (CsCl) solution (0.5%, by weight) in an ammonium acetate buffer(0.02M). The procedure in these cases was to place 150 μl of thesolution into a wedge filter of differing molecular weight cut-off(MWCO). The MWCO used were 30K, 50K and 100K Dalton. The filter was thencentrifuged and the samples were analyzed in the GEMMA. As shown inTable 5, the wedge filters all concentrated the MS2 solution, i.e. thecounts increased as the solution size decreased. Even with a subsequentaddition of buffer and re-centrifugation, the solutions continue toconcentrate.

The same solution (CsCl 0.5% +1×10¹¹ pfu/ml MS2) was then placed into a1M Dalton centrifuge filter and spun. The first concentration shows anincrease from 150 counts to 350 counts in the sample. The solutionvolume decreasing, from 1000 to 100 μl, should increase the countsmeasured. The subsequent wash and re-centrifugation should show anincrease in MS2 counts. However, the counts for the washed sample areeven lower. The conclusion from the filtration with the 1M MWCO filteris that the MS2 bacteriophage is able to pass through the filter and isnot retained.

TABLE 5 Filtration of MS2 plus CsCl Solutions Filter +1 MWCO Volume WashVolume Sample (Daltons) Counts (μl) (counts) (μl) CsCl 0.5% + 1 × 10¹¹None 150 150 MS2, DPG CsCl 0.5% + 1 × 10¹¹ 30K 2500 25 4500 35 MS2, DPGCsCl 0.5% + 1 × 10¹¹ 50K 2000 20 3000 25 MS2, DPG CsCl 0.5% + 1 × 10¹¹100K 9000 15 5000 10 MS2, DPG (+5 buffer) CsCl 0.5% + 1 × 10¹¹ 1M 350100 75 50 MS2, DPG centrifuge

To actually determine if the MS2 is passing through the centrifugefilters, the filtrate should be analyzed. A separate sample of 1×10¹²pfu/ml MS2 (DPG ultrafiltration cleaned, mixed media sample) wasfiltered with the 1M centrifuge filters. As shown in FIG. 18, the /MS2passed through the filter and was deposited in the filtrate. Table 6presents the numerical counts from the GEMMA analysis of the retentate,after one wash cycle, and the filtrate from the 1M centrifugation of thesample.

TABLE 6 Filtration of MS2 Solution after Ultrafiltration ProcessingFilter +1 MWCO GEMMA Volume Wash Volume Sample (Daltons) Counts (μl)(counts) (μl) DPG MS2 Mixed none 5000 100 Media UF Mod 1 DPG MS2 Mixed1M 75 100 Media UF Mod 1 centrifuge Retentate DPG MS2 Mixed 1M 3,500 150Media UF Mod 1 centrifuge Filtrate

To determine if there was any interference from the CsCl during thefiltration with the 1M filters, a solution of MS2 was prepared at aconcentration of 1×10¹¹ pfu/ml by dilution in the ammonium acetatebuffer only. The sample was prepared from a stock solution obtained fromthe Life Sciences Division of Dugway Proving Ground (DPG). The MS2solution was then centrifuged in the 1M centrifuge filter. As shown inTable 7, the plain MS2 solution also passed through the 1M filterapparatus with the loss of virus material. The CsCl does not appear toaffect the loss of virus material by its presence in the filtrationsolution.

TABLE 7 Filtration of Pure MS2 Solutions Filter MWCO GEMMA Volume Sample(Daltons) Counts (μl) 1 × 10¹¹ MS2, DPG None 600 100 1 × 10¹¹ MS2, DPG 1M 65 100 Retentate centrifuge

Another type of filtration is the cross flow or tangential flowtechnique. The solution is pumped through a hollow fiber that isdesigned to allow the passage of differing MWCO materials, depending onthe filter installed. A flow restriction at the exit from the fiberbundle develops a pressure differential that forces the filtrate throughthe fiber and concentrates the feed solution, as shown in FIGS. 9 and10.

The first sample prepared for filtration was a CsCl (0.05%, by weight)solution with 3×1011 pfu/ml MS2 added into the ammonium acetate buffer.The ultrafiltration parameters for this solution are shown in Table 8.As shown in Table 9, the sample volume was concentrated from 1000 to 100μl, but the counts dropped from 3200 to 25. This drop in counts showsthat the cross flow filter, at a MWCO of 750K Dalton, is allowing thevirus to pass through the hollow fiber.

TABLE 8 Cross Flow Parameters for CsCl (0.05%) plus MS2 (3 × 10¹¹)Sample volume-initial 1 ml Pump speed 2 Transducer pressure 15 psigTotal buffer wash volume 40 ml Sample volume-final 0.1 ml MWCO of module750K

TABLE 9 Cross Flow Filtration of CsCl (0.05%) plus MS2 (3 × 10¹¹) FilterMWCO GEMMA Volume Sample (Daltons) Counts (μl) CsCl 0.05% + 3 × 10¹¹MS2, DPG None 3200 1000 CsCl 0.05% + 3 × 10¹¹ MS2, DPG UF Mod1 25 100Retentate 750K

The second sample tested, a CsCl solution (2.5%, by weight) plus 5×10¹¹pfu/ml MS2 in ammonium acetate buffer, was processed through the crossflow filtration apparatus with a filter of 500K MWCO. The parameters forthe ultrafiltration processing of the solution are shown in Table 10.Although the sample volume was concentrated by half, the counts remainedconstant, as shown in Table 11. It appears that the MS2 virus is alsopassing through the 500K filter, although at a slower rate than the 750Kfilter.

TABLE 10 Cross Flow Parameters for CsCl (2.5%) plus MS2 (5 × 10¹¹)Sample volume-initial 1 ml Pump speed 2 Transducer pressure 15 psigTotal buffer wash volume 30 ml Sample volume-final 0.5 ml MWCO of module500K

TABLE 11 Cross Flow Filtration of CsCl (2.5%) plus MS2 (5 × 10¹¹) FilterMWCO GEMMA Volume Sample (Daltons) Counts (μl) CsCl 2.5% + 5 × 10¹¹ MS2,DPG None 800 1000 CsCl 2.5% + 5 × 10¹¹ MS2, DPG UF Mod1 750 500Retentate 500K

To test the lower limit of MWCO for a MS2 bacteriophage, a centrifugefilter of 300K MWCO was obtained. It appears from Table 1 that thefilters up to 100K MWCO do not allow the passage of MS2 through thefilter medium. The 300K filter was loaded with 100 μl, diluted to 1 mlin ammonium acetate buffer, of a 1×10¹¹ pfu/ml MS2 sample from DPG. Thesample was centrifuged and the retentate analyzed. As shown in FIG. 19,the MS2 is at least partially retained in the 300K filter.

To determine the amount, if any, of MS2 passing through the filter, a 1ml portion of the filtrate was concentrated in the 100K wedge filters.The final volume was reduced to 25 μl. As shown in FIG. 20, there wasMS2 present in the filtrate from the 300K centrifuge filtration. Itwould appear that the MS2 is able to pass through MWCO filters as smallas 300K. The MS2 does not appear to pass through the 100K centrifugefilters.

A series of solutions of 1×10¹² pfu/ml of MS2 bacteriophage will befiltered with the cross flow apparatus with a 750K MWCO ultrafilterinstalled. All of the filtered solutions will include 1 ml of the 1×10¹²pfu/ml MS2 with various additions of ammonium acetate buffer solution.The additions of buffer will allow differing lengths of time offiltration, in the cross flow apparatus, while keeping the amount of MS2in the sample constant. However, the concentration of the MS2 will varydepending on the dilution factor in the starting sample. The sampleswill be processed in the cross flow apparatus until concentrated toapproximately the 1 ml volume of the 1×10¹² pfu/ml MS2 initial sample.Table 12 presents the filtration parameters for the cross flow apparatusfor this set of experiments. Table 13 shows the starting volumes,initial dilution's, final sample volume and subsequent GEMMA samplecount for the MS2 viral peak.

TABLE 12 Cross Flow Parameters for MS2 (1 × 10¹²) plus Variable VolumeAmmonium Acetate Buffer Solutions Sample volume-initial 1 ml MS2 +variable buffer volumes Pump speed 2 Transducer pressure 15 psig Totalbuffer wash volume Variable Sample volume-final 0.70–0.75 ml MWCO ofmodule 750K

TABLE 13 Dilution Amounts and GEMMA Analysis of Cross Flow Filtration ofMS2 Samples Final GEMMA Counts MS2 Start Ammonium Acetate Volume for MS2Peak Volume Dilution (ml) (ml) (avg. of 2 runs) 1 ml @ 1 × 10¹² 0 1.09255 pfu/ml 1 ml @ 1 × 10¹² 1 0.75 5164 pfu/ml 1 ml @ 1 × 10¹² 2 0.705280 pfu/ml 1 ml @ 1 × 10¹² 4 0.75 3239 pfu/ml 1 ml @ 1 × 10¹² 8 0.705284 pfu/ml 1 ml @ 1 × 10¹² 16 0.75 3549 pfu/ml 1 ml @ 1 × 10¹² 32 0.702830 pfu/ml

The final volume of the solutions processed through the cross flowapparatus is essentially equivalent. The solutions should thereforeexhibit the same count rate for MS2, as the initial amount of virus wasequal in all cases. The count rates are plotted in FIG. 21, and show alogarithmic decline as the dilutions were increased. The increaseddilution's lengthened the contact time with the cross flow filter andsubsequently increased the loss of the MS2 bacteriophage through thefilter medium.

B.2. In an analysis, the MS2 bacteriophage was able to pass through thefilters of MWCO of 300K and higher daltons and was retained on filtersof 100K and less. This result was not expected as the bacteriophage hasan approximate size of 2M daltons, and was expected to be retained onthe initial filter of 750K MWCO size tested. Collins, et al observed asimilar result1, in a report to Koch Membrane Systems, Inc. This studyshowed the retention of MS2 bacteriophage with MWCO filters of 100Kdaltons and smaller and the passage of MS2 through a 500K dalton filter.The variable dilution cross flow filtration analysis in this reportshows the logarithmic removal of the MS2 from the feed stream, as thesolutions were concentrated. The longer the MS2 solution was in contactwith the cross flow filter of 750K, the more MS2 was removed from thesolution. If the goal of cross flow filtration is to remove salts andother ionic entities, a smaller MWCO filter (such as a 100K) could beused and the MS2 would be retained. However to remove largermacromolecules from a sample of MS2 bacteriophage, a different approachwould be needed. A larger MWCO filter (macromolecule dependent) would beused to retain and concentrate the macromolecule while the MS2bacteriophage is removed in the filtrate stream. The filtrate streamcould then be processed separately with a 100K MWCO filter to retain andconcentrate the MS2 bacteriophage. The extra step would only add a shortperiod of time to an analysis, as the cross flow filtration process is afast and efficient filtration.

The MS2 bacteriophage passed through 1M, 750K, 500K and 300K Daltonfilters. The phage was retained on the 100K Dalton centrifuge filter.The rate of virus passage is dependent upon back pressure for thetangential flow filters and on gravitational pressure for the centrifugefilters. Variable dilutions with cross flow filtration apparatus and a750K MWCO filter appear to produce a logarithmic removal of the MS2during filtration. Implications are clear that a better understanding ofmolecular weight cut off (MWCO) and how pore sizes are determined andreported need to be further investigated.

C. Characterization of MS2 Bacteriophage

A sample of MS2 bacteriophage provided by the Life Sciences Division atDugway Proving Ground (DPG) was analyzed and characterized. This samplewas 2 ml of purified MS2 bacteriophage at a concentration 1×10¹⁴ plaqueforming units (pfu)/ml or 10.2 mg protein/ml. This highly purifiedsample is from Lot #98110.

The MS2 sample was analyzed using the IVDS instrument or more directlythe Gas-phase Electrophoretic Mobility Molecular Analyzer (GEMMA)detector which is one stage of the IVDS instrument. The high purity MS2sample, with 1×10¹⁴ pfu/ml (hereafter described as DPM14) was analyzed.The sample of DPM14 was placed neat into the GEMMA analyzer and theresults are shown in FIG. 22. The graph shows a very high virus count(over 150,000 counts) as well as other features. MS2 is nominally 24–26nm in size and this is illustrated in FIG. 22. In fact, the sample asreceived was difficult to aspirate through the capillary delivery systemin the GEMMA.

The size range of 24–26 nm is the expected size for a MS2 bacteriophage.When the difficulty of sampling the neat MS2 sample became apparent, thesample DPM14 was then serially diluted to produce a number of lowerconcentration samples. That is, an aliquot of DPM14 was diluted 10 foldto produce a sample of MS2 at a concentration of 1×10¹³ pfu/ml. Thissample was named DPM13. The dilutions were all made with a 0.02Msolution of ammonium acetate (pH˜10), which is required for theelectrospray unit. The pH was adjusted to keep the virus from breakingdown into its component subunits. Sample DPM13 was then diluted 10 fold,and likewise for the following dilutions. Table 14 lists the samplesthat were produced by serially dilution of the original sample.

TABLE 14 Serial Dilution Samples of MS2 DPM13 1 × 10¹³ pfu/ml DPM12 1 ×10¹² pfu/ml DPM11 1 × 10¹¹ pfu/ml DPM10 1 × 10¹⁰ pfu/ml DPM9 1 × 10⁹pfu/ml DPM8 1 × 10⁸ pfu/ml

FIGS. 23–28 show the resultant GEMMA analysis of the serially dilutedMS2 samples. The counts for the serial dilutions were tabulated and areshown in Table 15.

TABLE 15 IVDS Physical Counts for MS2 Samples Counts in Size Range MS2Sample 25.946 nm 25.029 nm 24.144 nm 23.291 nm 22.468 nm DPM8  1 DPM9  25 3 DPM10 17 88 52 DPM11 146 929 541 78 DPM12 148 3613 12582 5174 255DPM13 15216 57624 65021 16893 1664 DPM14 96995 157461 150886 65389 8347

The GEMMA detector easily detects MS2 bacteriophage. The virus isconsistently detected in the range of 22 to 26 nm. The GEMMA scans alsoshow very low backgrounds away from the MS2 peaks. The action ofserially diluting the MS2 did not affect the stability of thebacteriophage in solution. In fact, the addition of ammonium acetatebuffer to produce dilutions reduced the background counts. The GEMMAscans of buffer solutions show very low counts, as ammonium acetate isnearly invisible to the detector.

The count rates for the various concentrations of MS2 were tabulated inTable 16. A comparison of the multiplication factor from sample tosample was also tabulated in the table. The lower concentrations displaya fairly consistent multiplier and are consistent with the targetdilutions. As the concentrations increase, the multiplier appears todecrease in magnitude. As was noted above, the as received sample,DPM14, was difficult to aspirate into the GEMMA detector. This sample isvery concentrated and this appears to interfere with the analysis. Thereduction in the multiplier may be caused by the agglomeration ofparticles as they flow through the Condensate Particle Counter (CPC) inthe GEMMA unit. This agglomeration would lower the amount of particlescounted and reduce the multiplier. It would appear that a count rateover 100,000 counts in a few adjacent channels, with a virus in thissize range of 25 nm, is approaching an upper limit to concentrationsthat can be analyzed in the detector. This is easily remedied by simplydiluting a sample to less than 100,000 counts in adjacent channels.

TABLE 16 Numerical Analysis of MS2 Peak Count Information MS2 Sum ofsize Multiplier from Sample range sample to sample DPM8  1 — DPM9  1010.0 DPM10 157 15.7 DPM11 1694 10.8 DPM12 21772 12.9 DPM13 156418  7.2DPM14 479078  3.1

The actual sensitivity of the GEMMA detector was not in question in thisstudy. The presented solution to the detector can be furtherconcentrated to allow for the analysis of samples that appear to be toodilute. The sample DPM8 could be concentrated from one ml, the originalvolume, to 10 μl. This would then present the GEMMA detector with asample that would generate a graph with ˜100 counts in a scan. Thenumber of viruses that can be detected by the GEMMA is very low, on theorder of 10 viruses, and therefore the ability to detect viruses is onlya function of the presented solution concentration. A further examplewas a simple experiment where a few thousand viruses were measured into500 ml of water. The water sample was concentrated through theUltrafilter unit and nearly 800 viruses were counted by the GEMMA. Thelimiting factor for analysis is the ability to further concentrate aliquid solution while still being able to effectively handle thesolution without losing it due the handling problems associated withtiny volumes.

The sample of MS2 bacteriophage received from the Life Sciences Divisionat Dugway Proving Ground was a very pure and concentrated sample. Noother viruses were detected. The sample responded well to serialdilutions and was stable in the ammonium acetate buffer. This techniqueis a simple method to test the purity of any virus preparation since theIVDS instrument is not limited to any particular virus.

It should be understood that the foregoing summary, detaileddescription, and drawings of the invention are not intended to belimiting, but are only exemplary of the inventive features which aredefined in the claims.

1. An apparatus for detecting the presence of submicron sized particleshaving a size range of from greater than 350 nanometers to about 1000nanometers in a sample taken from the environment, comprising: (a) acollecting means for collecting a sample from the environment; (b) meansfor purifying and concentrating the submicron particles in the sample bypurifying and concentrating the particles based on size, the purifyingand concentrating means including a means for connecting the collectingmeans to the purifying and concentrating means for transferring thesample from the collecting means to the means for purifying andconcentrating the particles; and (c) means for detecting the purifiedand concentrated particles, wherein the detecting means comprises: anelectrospray assembly which receives the output from the purifying andconcentrating means for placing a charge on the purified andconcentrated particles under the influence of an electric field, adifferential mobility analyzer which receives the output from theelectrospray assembly for separating the charged particles according tosize, and a condensation particle device for counting the number ofsized particles received from the differential mobility analyzer.
 2. Theapparatus of claim 1, wherein the collecting means comprises anultracentrifuge for density-gradient ultracentifugation of the sample sothat the particles are banded according to density.
 3. The apparatus ofclaim 1, wherein the collecting means comprises a collector having meansfor liquid scrubbing a collected fluid sample of aerosol and gaseousmaterials containing the particles and a means for reducing the size ofsolid materials in the fluid sample.
 4. The apparatus of claim 1,wherein the collecting means comprises a liquid sample collector.
 5. Theapparatus of claim 1, wherein the collecting means collects samples ofairborne aggregates which contain the particles and wherein theaggregates have sizes in the range of about 2–10 microns.
 6. Theapparatus of claim 1, further comprising conduit means connected to thecollecting means and the means for detecting the submicron sizedparticles for conveying the fluid sample from the collecting means tothe means for detecting the submicron sized particles.
 7. The apparatusof claim 1, wherein the submicron sized particles are selected from thegroup comprising viruses, prions, viral subunits, viral cores ofdelipidated viruses, plant viruses, standard particles used forcalibrating equipment, coated particles, spherical particles,metallic-core shelled particles, polymers, fluorescent microspheres,powders, nanoclusters, particles produced as a result of manufacturingprocesses, and portions of bacteria.
 8. The apparatus of claim 1,further comprising a first conduit means connected to the collectingmeans and the means for purifying and concentrating the submicronparticles for conveying the sample from the collecting means to themeans for purifying and concentrating the submicron sized particles. 9.The apparatus of claim 8, further comprising a second conduit meansconnected to the means for purifying and concentrating the submicronsized particles and the means for detecting the purified andconcentrated particles for conveying the purified and concentratedsample to the means for detecting the purified and concentratedparticles.
 10. The apparatus of claim 1, wherein the means for purifyingand concentrating the submicron sized particles comprises a filterapparatus.
 11. An apparatus for detecting the presence of submicron sizeparticles having a size range of from greater than 350 nanometers toabout 1000 nanometers in a sample taken from the environment,comprising: (a) a collecting means for collecting a sample from theenvironment; (b) filter means connected to the collecting means forseparating the particles in the collected sample based on the size ofthe particles; and (c) detecting means connected to the filter means fordetecting the separated particles, the detecting means comprising: anelectrospray assembly for receiving the separated particles and forplacing a charge on the separated particles, a differential mobilityanalyzer which receives the output from the electrospray assembly forseparating the charged particles based on the size of the chargedparticles, and a condensation particle device for counting the number ofseparated charged particles received from the differential mobilityanalyzer.
 12. An apparatus for detecting the presence of submicron sizeparticles having a size range of from greater than 350 nanometers toabout 1000 nanometers in a sample taken from the environment,comprising: (a) a collecting means for collecting a sample from theenvironment; (b) means for concentrating the submicron size particles inthe sample; and (c) detecting means connected to the concentrating meansfor detecting the concentrated submicron size particles, the detectingmeans comprising: an electrospray assembly for receiving theconcentrated submicron size particles and for placing a charge on theconcentrated submicron size particles introduced into the electrosprayassembly, a differential mobility analyzer which receives the outputfrom the electrospray assembly for separating the charged submicron sizeparticles according to the size of the charged submicron size particles,and a condensation particle device for counting the number of separatedsubmicron size particles received from the differential mobilityanalyzer.
 13. The apparatus according to claim 12, where the submicronsize particles are selected from the group comprising viruses, prions,viral subunits, viral cores of delipidated viruses, plant viruses,standard particles used for calibrating equipment, coated particles,spherical particles, metallic-core shelled particles, polymers,fluorescent microspheres. powders, nanoclusters, particles produced as aresult of manufacturing processes, and portions of bacteria.
 14. Anapparatus for detecting the presence of submicron size particles havinga size range of from greater than 350 nanometers to about 1000nanometers in a sample taken from the environment, comprising: (a) acollecting means for collecting a sample from the environment; (b) meansfor purifying the submicron size particles in the sample; and (c)detecting means connected to the concentrating means for detecting thepurified submicron size particles, the detecting means comprising: anelectrospray assembly for receiving the purified submicron sizeparticles and for placing a charge on the purified submicron sizeparticles introduced into the electrospray assembly, a differentialmobility analyzer which receives the output from the electrosprayassembly for separating the charged purified submicron size particlesaccording to the size of the charged purified submicron size particles,and a condensation particle device for counting the number of separatedcharged purified submicron size particles received from the differentialmobility analyzer.
 15. An apparatus for detecting the presence ofsubmicron sized particles having a size range of from greater than 350nanometers to about 1000 nanometers m a sample taken from theenvironment, comprising: (a) a collecting means for collecting a samplecontaining the particles from the environment; (b) means for separatingthe particles in the collected sample by separating the particles basedon size; (c) means for detecting the separated particles, wherein thedetecting means comprises: an electrospray assembly for receiving theseparated particles and for placing a charge on the separated particlesintroduced into the electrospray assembly, a differential mobilityanalyzer which receives the output from the electrospray assembly forseparating the charged particles according to the size of the chargedparticles, and a condensation particle device for counting the number ofseparated particles received from the differential mobility analyzer;and (d) valve means connected to the collecting means, the separatingmeans, and the detecting means, the valve means includes a means forselectively feeding the collected sample containing the particles eitherto the separating means or the detecting means.
 16. The apparatus ofclaim 15, wherein the collecting means comprises an ultracentrifugewhere the particles are banded according to density by density-gradientultracentifugation.
 17. A method for detecting the presence of submicronsized particles having a size range of from greater than 350 nanometersto about 1000 nanometers in a sample taken from the environment,comprising: (a) collecting a sample from the environment; (b) separatingthe submicron size particles in the sample based on the size of thesubmicron size particles; and (c) detecting the separated particles byplacing a charge on the separated particles, separating the chargedsubmicron size particles based on the size of the charged submicron sizeparticles; and counting the number of separated charged submicron sizeparticles.
 18. The apparatus according to claim 17, where the submicronsize particles are selected from the group comprising viruses, prions,viral subunits, viral cores of delipidated viruses, plant viruses,standard particles used for calibrating equipment, coated particles,spherical particles, metallic-core shelled particles, polymers,fluorescent microspheres, powders, nanoclusters, particles produced as aresult of manufacturing processes, and portions of bacteria.
 19. Amethod for detecting the presence of submicron sized particles having asize range of from greater than 350 nanometers to about 1.000 nanometersin a sample token from the environment, comprising: (a) collecting asample containing the particles from the environment; (b) purifying anticoncentrating the particles in the sample based on size; and (c)detecting the purified and concentrated particles with a detecting meanscomprising an electrospray assembly, the assembly having an electrospraycapillary which receives the output from the purifying and concentratingmeans, a differential mobility analyzer which receives the output fromthe capillary, and a condensation particle device for counting thenumber of purified and concentrated particles that pass through thedifferential mobility analyzer.
 20. The method of claim 19, wherein theliquid scrubbing step includes injecting water into the collectedaerosol and gaseous materials containing the particles and homogenizingthe liquid scrubbed aerosol and gaseous materials.
 21. The method ofclaim 19, wherein the collecting step comprises collecting a liquidsample in a container.
 22. The method of claim 19, wherein the purifyingand concentrating step comprises filtering the collected sample.
 23. Amethod for detecting the presence of submicron sized particles having asize range of from greater than 350 nanometers to about 1000 nanometersin a sample taken from the environment, comprising: (a) collecting asample from the environment; (b) concentrating the particles in thesample based on the size of the particles; and (c) detecting theconcentrated particles by placing a charge on the concentratedparticles, separating the charged particles based on the size of thecharged particles; and counting the number of separated chargedparticles.
 24. A method for detecting the presence of submicron sizedparticles having a size range of from greater than 350 nanometers toabout 1000 nanometers in a sample taken from the environment,comprising: (a) collecting a sample from the environment; (b) purifyingthe particles in the sample based on the size of the particles in thesample; and (c) detecting the purified particles by placing a charge onthe purified particles, separating the charged particles based on thesize of the charged particles; and counting the number of separatedcharged particles.
 25. An apparatus for detecting the presence ofdifferent size groups of submicron sized particles having a size rangeof from greater than 350 nanometers to about 1000 nanometers in a sampletaken from the environment, comprising: (a) a collecting means forcollecting a sample from the environment; (b) means for detecting theparticles in the collected sample, the detecting means comprising: anelectrospray assembly having an electrospray capillary which receivesthe collected sample from the collecting means, a differential mobilityanalyzer which receives the output from the electrospray; and acondensation particle counter means for counting the number of particlesin the collected sample.
 26. The apparatus according to claim 25, wherethe submicron size particles are selected from the group comprisingviruses, prions, viral subunits, viral cores of delipidated viruses,plant viruses, standard particles used for calibrating equipment, coatedparticles, spherical particles, metallic-core shelled particles,polymers, fluorescent microspheres, powders, nanoclusters, particlesproduced as a result of manufacturing processes, and portions ofbacteria.
 27. The apparatus of claim 26, wherein the collecting meanscomprises a liquid sample collector.
 28. The apparatus of claim 26,further comprising a calibration means connected to the collecting meansfor adding a calibration material of known size and concentration to thecollected sample for including in the output of the condensationparticle counter means an output of known size and concentration forreference with the size and concentration of the particles that arecounted.
 29. An apparatus for detecting the presence of different sizegroups of submicron sized particles having a size range of from greaterthan 350 nanometers to about 1000 nanometers in a sample taken from theenvironment, comprising: (a) a collecting means for collecting a samplecontaining the particles from the environment; and (b) detecting meansconnected to the collecting means for detecting the particles in thecollected sample, the detecting means comprising a means for placing acharge on the particles, a means for separating the charged particlesbased on the size of the particles and a means for counting the numberof separated particles in the collected sample.
 30. The apparatus ofclaim 29, further comprising a calibration means connected to thecollecting means for adding a calibration material of known size andconcentration to the collected sample for including in the countednumber of separated particles in the collected sample the calibrationmaterial of known size and concentration.
 31. A method for detecting thepresence of submicron size particles having a size range of from greaterthan 350 nanometers to about 1000 nanometers in a sample taken from theenvironment, comprising the steps of: (a) collecting a fluid samplecontaining submicron size particles selected from the group comprisingviruses, prions, viral subunits, viral cores of delipidated viruses,plant viruses, standard particles used for calibrating equipment, coatedparticles, spherical particles, metallic-core shelled particles,polymers, fluorescent microspheres, powders, nanoclusters, particlesproduced as a result of manufacturing processes, and portions ofbacteria; (b) directing the collected fluid sample to an electrosprayassembly having an electrospray capillary for introducing droplets ofthe fluid sample containing the submicron size particles into theelectrospray assembly under the influence of an electric field; (c)directing the output from the electrospray assembly to a differentialmobility analyzer for separating the submicron size particles accordingto size; and (d) directing the separated submicron size particles fromthe differential mobility analyzer to a condensation particle counterfor counting the number of submicron size particles in the fluid sample.32. The method of claim 31, further comprising the step of adding abiomarker of known size and concentration to the collected sample forincluding in the output of the condensation particle counter an outputof known size and concentration for reference with the submicron sizeparticles that are counted in a sample.
 33. A method for detecting thepresence of submicron size particles having a size range of from greaterthan 350 nanometers to about 1000 nanometers in a sample taken from theenvironment, comprising the steps of: (a) collecting a sample containingthe submicron size particles; (b) detecting the submicron size particlesin the collected sample by placing a charge on the submicron sizeparticles, separating the charged submicron size particles based on thesize of the submicron size particles, and counting the number ofseparated submicron size particles in the sample.
 34. The method ofclaim 33 further comprising the step of adding biomarker particles ofknown size and concentration to the collected sample for including inthe counted submicron size particles the biomarker particles of knownsize and concentration.
 35. The method of claim 33 further comprisingthe step of adding calibration material of known size and concentrationto the collected sample for including in the counted submicron sizeparticles the calibration material of known size and concentration.